Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Molecular characterization of human clathrin

H T Pretorius, P K Nandi, R E Lippoldt

    Biochemistry
    |May 12, 1981
    PubMed
    Summary

    Native clathrin (8.1S) from coated vesicles readily polymerizes into structures resembling those in cells. This polymerization assay helps determine clathrin preparation quality and activity.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    Prostate cancer transmitted in an orthotopic heart transplant.

    JAMA·1997
    Same author

    Effects of myocardial viability assessment with positron emission tomography on clinical management and patient outcomes.

    Chinese medical journal·1996
    Same author

    Bone scintigraphy and multimodality imaging in bone neoplasia: strategies for imaging in the new health care climate.

    Seminars in nuclear medicine·1994
    Same author

    Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells.

    Blood·1992
    Same author

    Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

    The Journal of biological chemistry·1992
    Same author

    Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol.

    The Journal of biological chemistry·1992

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Background:

    • Clathrin is a major protein component of coated vesicles.
    • Understanding clathrin's native structure and polymerization is crucial for cell biology.

    Purpose of the Study:

    • To characterize the biophysical properties of native clathrin.
    • To investigate the self-assembly of clathrin into higher-order structures.
    • To develop an assay for clathrin activity.

    Main Methods:

    • Sedimentation velocity and equilibrium ultracentrifugation.
    • Circular dichroism spectroscopy.
    • Size exclusion chromatography.
    • Guanidine hydrochloride denaturation.
    • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

    Main Results:

    • Native clathrin (8.1S) has a molecular weight of approximately 610,000 Da and a high frictional ratio, indicating an extended conformation.
    • Circular dichroism revealed significant alpha-helical content.
    • Purified clathrin monomer (in guanidine hydrochloride) had a molecular weight of approximately 170,000 Da.
    • At pH 6.3, 8.1S clathrin self-associates into high molecular weight species (150S and 300S), mimicking in-vivo coated vesicle structures.
    • Sedimentation analysis allows quantification of polymerizable clathrin, serving as an activity assay.

    Conclusions:

    • The 8.1S clathrin species is the native, polymerizable form.
    • Clathrin's self-assembly properties are fundamental to coated vesicle formation.
    • The described ultracentrifugation method provides a reliable assay for clathrin preparation quality and activity.

    Related Experiment Videos